Our article with the Henriques and Mercer labs is now out in Nature Methods. Previously posted on bioRxiv, it proposes a new metric to measure the quality of super-resolution images called NanoJ-SQUIRREL (Super-resolution Quantitative Image Rating and Reporting of Error Locations). Simply put, it compares the image to a reference diffraction-limited image, allowing to detect artefacts and missing features in the super-resolved image.
We used SQUIRREL to determine when to stop a STORM acquisition, showing that the longest acquisition was not always the best. In this case of imaging actin rings along axons labeled with phalloïdin, the quality of the STORM image as measured by SQUIRREL peaks after 30,000 frames and slightly drops when reaching 60,000 frames:
Another application of SQUIRREL is to optimize a DNA-PAINT experiment. In DNA-PAINT imaging, the density of the blinking events can be tuned by varying the concentration of the imager strand. We imaged clathrin-coated pits in a glial cell and the blinking sequence were processed with different algorithms. Each algorithm had a specific optimal blinking density (and thus imager strand concentration): SRRF was better with a denser blinking sequence, while pure localization algorithms such as MLE or CoM require a sparser acquisition sequence:
There are a lot more applications in the paper and its hefty supplementary file, go check it! Then try it thanks to the easy to use, open-source plugin for the ImageJ/Fiji plugin software.
Finally, this project is a striking example of open science in action. I (Christophe) met and started to collaborate with Ricardo and Jason on Twitter; the manuscript was posted on bioRxiv, where it got feedback from beta-testers and caught the attention of editors, before being accepted for publication six months later. Congrats to Siân, David, Caron, Pedro, and everyone!
Our collaboration with the lab of Jim Salzer (NYU) is just out. We visualized the association of phospho-myosin light chain (pMLC, an activator of the contractile myosin-II) with actin rings along the axon initial segment (AIS). This was done using two-color STORM. Moreover, acute treatment with KCL (mimicking elevated activity) resulted in a disappearance of the phospho-MLC signal before the disorganization of the actin rings. This suggests that myosin-II contractility has a role in setting the AIS shape and position along the axon, and that release of this contractility could be a key remodelling step for the activity-dependant plasticity of the AIS.
Our review on the nano-architecture of the axonal cytoskeleton is out today on the Nature Reviews Neuroscience website. This was a lot of work and a lot of fun to write with Pankaj Dubey and Subhojit Roy. We tried to provide an up-to-date view that discusses recent findings such as the various axonal actin structures visualized along the axon by STORM. We also wanted to highlight the classic EM works that shaped how we think about the axonal cytoskeleton. So it’s chock-full of recent references with fancy techniques, but also beautiful classic papers. We hope it will be a pleasant reading for all!
In collaboration with Matt Rasband’s lab in Houston, we characterized the α-spectrin that is present along axons at the axon initial segment (AIS) and nodes of Ranvier. This work is out today as two back-to-back paper just pre-published on the Journal of Neuroscience website, here and here. Spectrins are tetramers of two α and two β subunits. It is known that the β-spectrin form at the AIS and nodes is the ßIV-spectrin since 2000, but the identity of the α subunit was unknown. In the axon, spectrins binds submembrane actin rings regularly spaced every 190 nm. As this is just below the resolution limit of conventional fluorescence microscopy (~200 nm), the resulting periodic scaffold is only visible using super-resolutive techniques such as STORM.
The first paper: “αII spectrin forms a periodic cytoskeleton at the axon initial segment and is required for nervous system function” focuses on the identification of αII-spectrin as the ßIV-spectrin partner at the AIS, and the consequences of αII-spectrin depletion in CNS-specific knockout mice. We used super-resolution microscopy to show that αII-spectrin is integrated in the AIS periodic actin/spectrin scaffold that supports the axonal plasma membrane. With the αII-spectrin antibody we used, the periodicity is seen as double bands every 190 nm by STORM. When using 2-color DNA-PAINT to image αII-spectrin together with ßIV-spectrin, the doublet of αII-spectrin labeling appears on both sides of the ßIV-spectrin bands, resolving the organization of the spectrin tetramers in situ. We also showed by STORM that the periodic actin/spectrin complex is disorganized in αII-spectrin-depleted neurons.
The second paper: “An αII spectrin based cytoskeleton protects large diameter Myelinated axons from degeneration” focuses on αII-spectrin in the PNS and nodes of Ranvier. In C. elegans mutants, the submembrane spectrin scaffold is necessary for the mechanical resistance of axons. Here, an αII-spectrin knockout mouse specific to peripheral sensory neurons was used to demonstrate this for in a vertebrate. Using STORM, we showed that loss of αII-spectrin causes a disorganization of the periodic scaffold at and around nodes. This disorganization ultimately results in the degeneration of large-diameter peripheral axons lacking αII-spectrin.